Summary

Objective: To test a protocol, using conditions found on commercial
swine production units, for sanitation of 1:150 scale models of commercial
transport vehicles contaminated with porcine reproductive and respiratory syndrome
virus (PRRSV).

Methods: Model trailers were experimentally contaminated with PRRSV
MN 30-100 and either pressure-washed with cold water alone (Treatment 1) or
washed and then disinfected with modified potassium monopersulfate (Treatment
2), quaternary ammonium chloride (Treatment 3), or a phenolic product (Treatment
4), each applied using a hydrofoamer. In Phase One, the presence of PRRSV RNA
was evaluated by polymerase chain reaction (PCR) testing of swabs collected
from the trailers' interiors immediately after washing and 30, 60, 90, and
120 minutes post treatment. In Phase Two, the presence of viable PRRSV was
evaluated by swine bioassay (injection of supernatants from PCR-positive swabs)
and housing pairs of sentinel pigs in treated trailers for 2 hours, beginning
90 minutes post treatment.

Implications: High-pressure washing of transport trailers, followed
by 90 to 120 minutes exposure to either modified potassium monopersulfate or
quaternary ammonium chloride disinfectants applied with a hydrofoamer is likely
to eliminate residual infectious PRRSV.

Keywords: swine, disinfectant,
porcine reproductive and respiratory virus, transport, vehicleSearch the AASV web site
for pages with similar keywords.

Received: April
25, 2005Accepted: July
7, 2005

Porcine reproductive and respiratory
syndrome virus (PRRSV) is a single- stranded enveloped RNA virus
classified in the order Nidovirales, family
Arteriviridae, and genus
Arterivirus.1 The disease caused by PRRSV, known as porcine
reproductive and respiratory syndrome (PRRS), has proven to be very difficult to control
consistently over time and across farms. A key component to successful control of
PRRS is preventing spread of the virus within and between farms.
Transmission of PRRSV can occur through a number of reported
routes, including infected pigs, semen, contaminated fomites, insects,
avian species, and aerosols.2-8 Another potential route
of transmission between farms may be the livestock transport
vehicle.9 In today's modern pig industry, the application of
multi-site production technology has resulted in more movement of pigs and
greater distances between sites and to slaughter.
Therefore, pig transport has become a highly important risk factor for transmission of
PRRSV. In support of this hypothesis, previously published
reports10-12 have demonstrated how motorized vehicles can
mechanically transport PRRSV over distances of 50
km, and specific assessments of the role of the transport vehicle in transmission
of PRRSV have been conducted. In a recent
study,12 1:150 scale models of
weaned-pig trailers were used to enhance study
power. The materials and designs used in these models were similar to those used in
commercial transport vehicles, and the models provided for an animal density equal
to that in a weaned-pig trailer capable of transporting 300 pigs. Under the
conditions of that study,12 it was
demonstrated that PRRSV-naive swine could become infected with PRRSV through contact
with the contaminated interiors of the transport models. It was also determined that
the concentration of PRRSV required to infect naive sentinel pigs was 1
x 103 median tissue culture infectious doses
(TCID50) and that allowing the trailer to dry for 8
hours after washing effectively prevented infection in 10 of 10
replicates.12 However, discussion of these results with
veterinarians working in large commercial systems
indicated that sanitation programs requiring time periods > 2 hours limit the
cost-effective use of trailers. Furthermore,
accessibility to hot water (80°C) for washing
was limited, and use of a low-pressure foaming system was a common method of
applying disinfectant. Foam provided an effective vehicle to carry the disinfectant
to the target surface and a means to hold it there in the short term. This technique
has the added advantage of allowing the operator to see where the disinfectant
has been applied.

Despite a growing interest in the use of foaming to apply disinfectants, there
is little scientific evidence demonstrating its efficacy against PRRSV. While
previous work had evaluated the ability of commercially available disinfectants to
sanitize PRRSV-positive transport vehicles, the
disinfectants had been applied by fogging, not
foaming.13,14 Therefore, this study was conducted to test the principle of
foaming in a sanitation protocol designed for PRRSV-positive commercial transport
vehicles. To improve the authenticity of the study, the protocol incorporated
several other methods frequently used in commercial swine systems, including cold water
for washing and rapid turn-around time of trailers (< 2 hours). Turn-around time
was defined as the time required to sanitize a contaminated trailer after animals
were unloaded.

Materials and methods

Trailer models

Figure 1: Four 1:150 models of full-size weaned-pig
trailers were designed to allow for an equivalent animal density within
the model trailer (2.5-kg pigs at 0.07 m2 per pig) compared
to a full-sized trailer loaded to capacity (300 five-kg pigs). Dimensions
are provided in Table 1.

To allow for multiple replications, the University of Minnesota Department
of Biosystems and Agricultural Engineering constructed four 1:150 scale models
(Figure 1) of full-size weaned-pig
trailers.12-14 This scale allowed for an animal density
within the model trailer (2.5-kg pigs at 0.07
m2 per pig) equivalent to that in a
full-sized weaned-pig trailer loaded to capacity
(300 five-kg pigs). As in full-size trailers, the frame, roof, and exterior sidewalls of
the model trailers were flat aluminium, the flooring was polished aluminium
tread-plate, and the interior walls were covered
with textured styrene and insulated with foil-coated Styrofoam. Each exterior
sidewall contained openings for proper
ventilation, and a locking door was available on
the end of each model. The dimensions of the full-size trailers and the models are
provided in Table 1.

Experimental design

The study was conducted in two phases at the University of Minnesota Swine
Disease Eradication Center (SDEC) research
farm. In Phase One (detection of PRRSV RNA), each trailer model was deliberately
contaminated with PRRSV, then assigned a specific disinfectant treatment. In
Phase Two, infectivity of trailers was determined by swine bioassay and by housing
naive sentinels in treated trailers.

Animal care and housing

All pigs were obtained from a source documented as PRRSV-naive on the basis of
10 years of clinical, diagnostic, and production data. All pigs were blood tested by
polymerase chain reaction (PCR) and ELISA to confirm PRRSV-naive status upon arrival
at the study site at 3 weeks of age.

Animals were cared for under the guidelines of the University of Minnesota Animal
Care and Use Committee, which approved the study protocol. Animals were housed in
commercial nursery pens with wire-mesh flooring and commercial feeders and waterers.
Heat lamps were provided as needed, and fresh air was delivered by a
negative-pressure power ventilation system. Animals
were observed twice daily to insure that a comfortable living environment was in place.

Contamination of trailer models with PRRSV

The mechanically ventilated nursery room (25
m3) used for the study was heated to 20°C. Trailer models were placed in
adjacent pens in the room (two trailers per pen). Trailer floors were covered with
wood shavings, emulating a common practice in the North American seed-stock
industry. As in the other investigations of PRRSV transmission by
transport,10-14 the strain of PRRSV employed was a field isolate
designated as MN 30-100.15 For each
replicate, PRRSV was prepared at a concentration
of 5 x 105 TCID50 in 5-mL aliquots of
minimum essential medium (MEM; Difco Laboratories, Detroit, Michigan).
Walls, ceilings, and floors of each trailer were
inoculated using a hand-operated, multi-use power mister (Chapin
Manufacturing, Batavia, New York). This high
concentration of PRRSV was selected to exceed the previously determined concentration
necessary to infect naive sentinel pigs housed in the model trailers, in order to
thoroughly test disinfectant
efficacy.12 After contamination, each trailer was assigned a
specific treatment and placed in a pen in the nursery area, with maximum separation of
the four pens housing the trailers during the treatment and intervention procedures.

Trailer washing and disinfectant treatment protocols

Treatments included washing with cold water alone (Treatment 1) and washing
and then disinfecting with one of three disinfectants: modified potassium
monopersulfate (Treatment 2); quaternary ammonium chloride (Treatment 3); and a phenolic
disinfectant (Treatment 4). Swabs were collected from the interior of each trailer
before and after treatment. A total of 20 replicates were conducted for each
treatment, allowing for detection of a 48% reduction
in the proportion of infected trailers at a target alpha level of 0.05 and an 80%
study power.

In an effort to replicate protocols used in the field for transport time and
sanitation of transport vehicles, data from an
international breeding stock company (Genetiporc, Alexandria, Minnesota) were used
throughout the study. This company sells breeding boars and gilts throughout North
America and Latin America and operates approximately 15 transport vehicles, delivers
approximately 1800 to 2000 truckloads of animals per year, and conducts
approximately 30 to 35 sanitation procedures per week.

The interiors of all trailers were manually scraped with a hand-held plastic scraper
to remove soiled bedding. To insure that mechanical transmission of PRRSV did
not occur between treated and control trailers, the blade of the scraper was
immersed in 70% ethanol, rinsed with sterile water,
and swabbed between trailers. Trailers were then washed for 72 seconds using a
commercial power washer (model TB5030A; American Made Cleaners, Beresford,
South Dakota) that provided cold water (20°C) delivered at a pressure of 3000 psi.
This wash time was extrapolated from the approximately 2-hour wash time for a
full-sized weaned-pig trailer (R. Witt,
Genetiporc, personal communication, April 2002).

Application of disinfectants

Bedding was removed and contaminated trailers were washed as described. No
disinfectant was applied to trailers assigned Treatment 1. Disinfectants were applied
to trailers assigned the other three treatments using a hydrofoamer (Hydro
Systems Company, Cincinnati, Ohio) attached to a garden hose. Foam was applied to the
interior of each trailer for 72 seconds, using a 1% solution of modified
potassium monopersulfate (Treatment 2), and 30
mL of disinfectant per 3840 mL of water for the quaternary ammonium chloride
product (Treatment 3) and the phenolic product (Treatment 4). For the control
protocol, trailers were sham-disinfected using saline in the
hydrofoamer.

Phase One: Detection of PRRSV RNA

Collection and testing of swabs. Before treatment (immediately after washing)
and 30, 60, 90, and 120 minutes post treatment, swabs were collected from the
interior of each trailer (floor, four walls, and ceiling, for a total of 0.14
cm2). For each trailer, a sterile swab (Dacron
fiber-tipped plastic applicator swabs; Fisher
Scientific Company, Hanover Park, Illinois) was moistened with MEM. Then, starting
from the left side of each surface and progressing across the surface in a rightward
direction, each swab was drawn over the walls,
floor, and ceiling using a zigzag pattern. Swabs were then placed in sterile plastic
tubes (Falcon, Franklin Lakes, New Jersey) containing 2 mL of MEM, and frozen at
-20°C. When all required samples had been collected, swabs were tested for PRRSV
RNA in duplicate using the TaqMan PCR assay (Perkin-Elmer Applied Biosystems,
Foster City, California).16 The reaction was
defined as "suspect" when one of the two PCR results was positive.

Phase Two: Infectivity of trailers

Swine bioassay. For this phase, a total
of 16 pigs were employed. To prepare the bioassay
samples,17 swabs in MEM were selected at the last sampling time when
samples were PCR-positive, supernatant was removed and pooled by treatment, and 20 mL
of MEM was added to each 20-sample pool. Each of the four treatment pools was
divided into 5-mL aliquots that were injected IM (four
pigs per treatment, 5 mL per pig). After injection, each pig was placed in
an individual pen in a room assigned to the treatment group. Nose-to-nose contact
was prevented. Biosecurity measures to prevent transmission of PRRSV between
rooms4,18 included changing boots, gloves, and
overalls between rooms, and 10-second immersion of boots in modified potassium
mono-persulfate boot baths upon entering each room. Blood samples were collected
from each pig before injection (Day 0) and on Days 7 and 14, and sera were tested
for PRRSV RNA by PCR and for PRRSV antibodies by the Idexx 2X-R ELISA
(Idexx Laboratories, Westbrook, Maine).

Sentinel exposure. Ninety minutes after completion of each trailer treatment
replicate, two PRRSV-naive sentinel pigs were placed in each treated trailer for a
2-hour "transport" period (eight pigs total).
This length of time was based on the mean time of 2 hours, reported by Genetiporc,
required for a shipment of pigs to leave Site 1 (breeding, gestation, and farrowing
farm) and arrive at Site 2 (nursery). After the 2-hour "transport" period, each sentinel
pig was removed from the trailer and housed as described for bioassay pigs. Blood
samples were collected from all sentinels before
the 2-hour transport period began (Day 0) and on Days 7 and 14 and tested as
described for the swine bioassay.

Controls

As validation that the methods used in treating the trailers did not
accidentally contaminate the models, 20 replicates of
a protocol control were conducted. For the purpose of this control, trailers were
sham-inoculated with saline and swabbed as described. Finally, after each replicate of
each treatment was completed, trailers were rewashed, hand-dried with disposable
paper towels, and swabs were again collected for PCR testing to determine whether
trailers were free of residual PRRSV RNA, in order to verify that each replicate
was an independent event.

Statistical analysis

All treatments were tested using a one-tailed chi-square model,
examining whether there was a significant
difference in the number of positive samples over time within treatments.

Results

High-pressure washing did not completely remove organic debris from the
trailers. Small amounts of residual bedding were visible in all trailers after the
72-second wash.

Phase Two: Detection of viable PRRSV

The results of the swine bioassay testing and the trailer infectivity assessments
are also summarized in Table 2. All swab supernatants from trailers treated with
modified potassium monopersulfate and quaternary ammonium chloride were negative
on swine bioassay. In contrast, infectious PRRSV was detected in one of
four samples collected from the sentinel pigs in trailers treated with the phenolic
disinfectant and from four of four samples from pigs exposed to trailers treated with no
disinfectant. Adverse side effects (irritation of the skin, abscess formation, swelling)
were not detected at the site of injection of the bioassay sample, nor did pigs
experience fever or loss of appetite.

Sentinels were infected in three of the four replicates for pigs housed in trailers
not treated with disinfectant (Treatment 1), but in no replicates for Treatments 2,
3, and 4 (Table 2).

Table 2: Results of polymerase chain reaction
(PCR), swine bioassay, and sentinel exposure testing after trailer models
contaminated with porcine reproductive and respiratory syndrome virus
(PRRSV) were pressure washed and either treated with a disinfectant or
not treated*

* In all four treatments, 1:150-scale trailers were washed with water
at 20°C, and disinfectants were applied with a hydrofoamer (1% peroxygen
and 30 mL disinfectant/3840 mL of water for the quaternary ammonium chloride
and phenolic products). One swab per treatment was collected from the
interior of each trailer for PCR testing (20 replicates per treatment).
For swine bioassay, four PRRSV-naive sentinel pigs per treatment were
injected with supernatant from PCR-positive swabs collected at the latest
post-treatment time when swabs were positive. For trailer infectivity,
two sentinel were confined in each trailer for a 2-hour period beginning
90 minutes post treatment (four replicates per treatment, eight pigs
total). Swine bioassay pigs and sentinel pigs were tested by serum PCR
for PRRSV and by ELISA for PRRSV antibody on the day of injection or
exposure, respectively, and 7 and 14 days later to determine a change
in PRRS infection status.

Results were defined as suspect when one of two duplicate PCR tests
was positive.

a Difference significant (P < .01) when compared
with result at previous sampling time (60 minutes).

b Difference not significant (P = .24) when compared
with result at previous sampling time (60 minutes).

Washing alone (Treatment 1) had no statistically significant effect over time,
with only a 10% decrease in number of positive samples at 120 minutes post
disinfection. For Treatments 2 through 4, there
were significantly fewer positive samples at contact times > 60 minutes compared to
contact times <= 60 minutes. At 90 minutes, all samples were negative, and this was
significantly less than at 60 minutes for all three disinfectants.

Discussion

The justification for this study was that scientific data on the efficacy of
foaming for decontamination of PRRSV-positive transport vehicles were not available,
and that this method of application of disinfectant was rapidly increasing in
North American production systems. Under the conditions of the study, washing with
cold water alone had little impact on eliminating PRRSV from trailer interiors,
supporting previously published
work.12-14 PRRS virus RNA was detected in 80 of 80
swabs (100%) collected immediately after the washing procedure across all
replicates. Also, despite the use of high
pressure, washing did not result in complete
removal of organic debris from the trailers, a frequent observation under
field conditions.

Results also indicated a difference in the performance of disinfectants tested in
this study compared to previously published
work.14 Samples from trailer models treated with modified
potassium monopersulfate or quaternary ammonium chloride were negative for PRRSV RNA
by PCR in all replicates at 90 and 120 minutes post treatment. No evidence of
infectious PRRSV was detected in trailers treated with these products, as
demonstrated by negative swine bioassay results and lack of seroconversion of sentinels
post exposure to treated trailers. These results differ from previously reported
results.14 One explanation may have been the use
of a different concentration of the quaternary ammonium chloride product in this
study (30 mL disinfectant per 3840 mL water) compared to the concentration of 15
mL disinfectant per 3840 mL water in the previous
study.14 In addition, different means of applying the disinfectant were
used (foaming and fogging).

In trailers treated with the phenolic product, PRRSV RNA was not detected at
90 minutes post treatment, but there were two suspect results 120 minutes post
treatment in this treatment group. At the
Minnesota Veterinary Diagnostic Laboratory, duplicate testing of each sample by PCR is
standard protocol, and suspect results are reported if one of the two tests is
positive. Again, these results differ from those
previously reported for this product.14
Possible explanations could again include the
difference in method of application (foaming and fogging), previously described
differences in the concentration of the product, or the difference in room temperature
under which the studies were conducted (20°C and 4°C). In contrast to results
for the modified potassium monopersulfate and quaternary ammonium chloride
treatments, one of four pooled swab supernatants from trailers treated with the
phenolic product was bioassay-positive, indicating the presence of infectious PRRSV;
however, sentinels housed in treated trailers were not infected. This finding
confirms the presence of infectious PRRSV in the trailer interior and suggests differences
in efficacy across the three products tested. However, sentinels housed in trailers
may have failed to become infected because of an insufficient quantity of PRRSV, an
inadequate number of replications conducted to detect the
frequency of this event, or the inability of the pigs to access the virus
due to the confines of the model.

This study contained several acknowledged limitations. Testing swabs by PCR
alone was a primary limitation, as it was not possible to determine whether viable
PRRSV was present in the trailer interior post
treatment. This was countered by use of confirmatory tests, ie, live-pig exposure to
treated trailers and swine bioassay to attempt to determine conclusively whether a
positive PCR result was indicative of live or dead virus. Multiple factors may have
contributed to negative PCR results, including the diagnostic sensitivity of the test,
degradation of viral RNA in the sample through prolonged contact with the
disinfectant, interference of the disinfectant with
the PCR assay, or a truly efficacious disinfectant not only rendering the virus
inactive but also degrading its nucleic acid. In
the authors' opinion, test sensitivity did not appear to be an issue, as the TaqMan
PCR assay is regularly able to detect PRRSV RNA in numerous samples, with a
reported level of detection of 0.01
TCID50 per PCR reaction.16 The possibility of
degradation of PRRSV RNA in swab samples secondary to prolonged contact with
disinfectant during storage did not appear to be a problem, as shown by the large
number of PCR-positive samples detected at 30 and 60 minutes post treatment across the
various treatments. It was not possible in this study to add an agent to neutralise the
disinfectant because of the potential virucidal effects of exogenous chemicals.
Therefore, swabs were stored at -20°C
immediately post collection to retard disinfectant
activity,19-21 and underwent RNA
extraction within 24 hours post collection, the
standard practice in our previous studies on this
subject.13,14 Potential interference of the individual disinfectants with the
PCR assay did not appear to be an issue. PRRS virus RNA was detected in all replicates
30 minutes after application of treatments, indicating that it was possible for
the TaqMan PCR assay to function properly in the presence of residual disinfectant.

Another acknowledged limitation of the study was inability to counteract the
impact of drying that naturally occurred during the sampling period of 120
minutes. Other acknowledged limitations include inability to use full-size trailers and
large loads of pigs. The models were scale models of weaned-pig trailers, and their
construction design does not mimic a trailer that transports market animals,
variables that certainly could impact the level of contamination in the trailer interior
and the ease of cleaning. It is not known if the high concentration of PRRSV used to
contaminate the trailers was representative of actual transport conditions. The entire
interior of the models was contaminated, and this may not be representative of field
conditions. It has been previously
determined12 that sentinel pigs can be
infected with PRRSV when these model trailers are contaminated with PRRSV
concentrations of 1 x 103
TCID50. Therefore, in order to aggressively test the efficacy of the
decontamination protocol, a high concentration of virus was selected as before. It must
also be noted that the size of the swab was not proportional to the size of the model
trailer and this may have impacted the recovery of PRRSV RNA. It was also not possible
to conduct the study using market-age animals or adult breeding swine.
Furthermore, although a relatively large number of
replicates were conducted in Phase One, it was not sufficient to predict the frequency
of the events recorded in the study. In addition, live animals were used in only
four replicates; due to this small sample size, no estimation can be made regarding the
frequency of the reported events. It was not possible to quantify the amount of
PRRSV RNA present in PCR-positive samples. Finally, the results of this study cannot
be directly extrapolated to other swine pathogens, such as transmissible
gastroenteritis virus or Mycoplasma
hyopneumoniae.

Despite its limitations, the study had considerable strength. It introduced use of
a hydrofoamer for sanitation of PRRSV-contaminated surfaces and provided
preliminary information on its efficacy against
PRRSV. The equipment was easy to use, and its ability to provide visual confirmation
of contact (ie, white foam) between the disinfectant and the surface will ensure
better and more accurate application of disinfectant in repeated commercial usage.
It showed that chemical sanitation using the modified potassium monopersulfate
and the quaternary ammonium chloride products produced good inactivation of
PRRSV within the target time when used with cold-water washing and application of
disinfectant via foaming, further enhancing understanding of PRRSV sanitation
protocols for commercial transport vehicles. While it was true that the trailer size
and pig numbers were small, the models provided for an animal density equivalent
to that of a full-size trailer. Furthermore, it would have been impossible to obtain
full-size trailer loads (200 to 300 pigs) for even a single replicate, much less to repeat
the study at any frequency. The use of industry standards for transport times and
wash-water temperature and pressure, as well as commonly used disinfecting products
and practices (ie, use of wood shavings) replicated real-world situations. One
additional factor not included in the study design
was use of detergents to facilitate removal of organic debris, and inclusion of such
products might have enhanced the results and may decrease the time required for
cleaning.

Implications

These results will enable swine producers and practitioners to
further understand and appreciate the merit of sanitizing livestock transport vehicles.

Disinfectants may differ in their efficacy against PRRSV.

Critical factors in sanitation program for PRRSV-contaminated
transport vehicles include selecting an
efficacious disinfectant, using it at the proper dilution rate and means of
application, and allowing for sufficient contact time.

Acknowledgements

The authors would like to thank Dr Anna-Maria Castiglia-Zanello and Mr
Paul Russell of Dupont Animal Health for technical assistance and financial support
for this study.